Neonatal stroke occurs more frequently in males than females across diverse ethnic backgrounds and nationalities. However, the sex steroid environment at the time of stroke does not completely explain the sexual dimorphism observed in stroke risk and outcome in children. Preliminary data indicates that sex differences in ischemic sensitivity also extends to the cellular level, as astrocytes and neurons from male neonatal rodents are more sensitive to oxygen-glucose deprivation (OGD), an in vitro model of ischemia, than cells from female neonatal rodents. Our preliminary data suggest that the sex-specific responses of astrocytes to OGD are linked to developmental reprogramming by testosterone during sexual brain differentiation. The central hypothesis is that the developing brain can assume either a male or female basal ischemic sensitivity, with males being more sensitive, and that testosterone-induced reprogramming of astrocytes during brain differentiation shapes the development of male rather than female cellular (astrocyte and neuronal) ischemic sensitivity in neonatal brain. This proposal is innovative in that it will link testosteroe exposure during brain development to sex differences in cellular ischemic sensitivity in neonatal brain via a novel astrocyte-specific mechanism involving microRNA 29 (miR-29) mediated regulation of de novo DNA methylation enzymes and subsequent epigenetic modifications and expression of genes responsible for shaping ischemic sensitivity of astrocytes directly and of neurons indirectly. To investigate this mechanism directly, we will use an innovative approach, whereby testosterone levels during brain sexual differentiation are altered in vivo in male and female rats and then sex-stratified cultures of astrocytes and neurons derived from these rats postnatally are studied in vitro separately and in co-cultures. Aim 1 will determine if increased ischemic sensitivity of male vs. female astrocytes and neurons is due to the effects of testosterone-induced reprogramming of astrocytes during brain sexual differentiation. Hypotheses are that manipulation of testosterone levels during brain sexual differentiation will reverse sex-specific sensitivity to OGD in astrocytes and neurons. Aim 2 will determine if testosterone-induced reprogramming of astrocytes during brain sexual differentiation increases cellular (astrocyte and neuronal) ischemic sensitivity by altering expression of the miR-29 family and its target genes, de novo DNA methyltransferases 3A and 3B (DNMT3A and 3B), in astrocytes. Hypotheses are that exposure to testosterone during brain differentiation leads to increased astrocyte and neuronal sensitivity to OGD via reduced miR-29 family expression and consequent increased DNMT3A and 3B expression in astrocytes. Lentiviral overexpression or knockdown of the miR-29 family will be used to link changes in the miR-29 family to molecular and ischemic outcomes in astrocytes and neurons. The proposed work is significant as it will characterize outcomes, epigenetic events, molecular pathways and cell-specific effectors underlying the development of cellular (astrocyte and neuronal) ischemic sensitivity in male vs. female neonatal brain. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it will address gaps in our knowledge regarding sex differences in neonatal stroke by determining if the sexual dimorphism observed in neonatal stroke is in part shaped by events early in development, specifically testosterone exposure during brain sexual differentiation. Our findings will characterize a potentially key factor in determining what aspects of cellular ischemic sensitivity in neonatal brain are modifiable vs. developmentally programmed as well as in predicting whether existing or new therapies are likely to be equally efficacious in male and female neonates. The proposed studies are also relevant to the mission of NINDS to reduce the burden of neurological diseases through research on the causes and treatment of neonatal stroke.